title: Diacylglycerol kinases terminate diacylglycerol signaling during the respiratory burst leading to heterogeneous phagosomal NADPH oxidase activation.
abstract: It is commonly assumed that all phagosomes have identical molecular composition. This assumption has remained largely unchallenged due to a paucity of methods to distinguish individual phagosomes. We devised an assay that extends the utility of nitro blue tetrazolium for detection and quantification of NAPDH oxidase (NOX) activity in individual phagosomes. Implementation of this assay revealed that in murine macrophages there is heterogeneity in the ability of individual phagosomes to generate superoxide, both between and within cells. To elucidate the molecular basis of the variability in NOX activation, we employed genetically encoded fluorescent biosensors to evaluate the uniformity in the distribution of phospholipid mediators of the oxidative response. Despite variability in superoxide generation, the distribution of phosphatidylinositol 3,4,5-trisphosphate, phosphatidylinositol 3-phosphate, and phosphatidic acid was nearly identical in all phagosomes. In contrast, diacylglycerol (DAG) was not generated uniformly across the phagosomal population, varying in a manner that directly mirrored superoxide production. Modulation of DAG levels suggested that NOX activation is precluded when phagosomes fail to reach a critical DAG concentration. In particular, forced expression of diacylglycerol kinase β abrogated DAG accumulation at the phagosome, leading to impaired respiratory burst. Conversely, pharmacological inhibition of DAG kinases or expression of an inactive diacylglycerol kinase β mutant increased the proportion of DAG-positive phagosomes, concomitantly potentiating phagosomal NOX activity. Our data suggest that diacylglycerol kinases limit the extent of NADPH oxidase activation, curtailing the production of potentially harmful reactive oxygen species. The resulting heterogeneity in phagosome responsiveness could enable the survival of a fraction of invading microorganisms.
authors.affiliation: Program in Cell Biology, Hospital for Sick Children, Toronto, Ontario M5G1X8, Canada.
authors.forename: Daniel
authors.initials: D
authors.lastname: Schlam
authors.forename: Michal
authors.initials: M
authors.lastname: Bohdanowicz
authors.forename: Alexandros
authors.initials: A
authors.lastname: Chatgilialoglu
authors.forename: Alexandros
authors.initials: A
authors.lastname: Chatilialoglu
authors.forename: Benjamin E
authors.initials: BE
authors.lastname: Steinberg
authors.forename: Takehiko
authors.initials: T
authors.lastname: Ueyama
authors.forename: Guangwei
authors.initials: G
authors.lastname: Du
authors.forename: Sergio
authors.initials: S
authors.lastname: Grinstein
authors.forename: Gregory D
authors.initials: GD
authors.lastname: Fairn
mesh_terms: D000818:Animals; D004075:Diglycerides / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation / Q000502:physiology; D006367:HeLa Cells; D006801:Humans; D008071:Lipoprotein Lipase / Q000235:genetics / Q000378:metabolism*; D008264:Macrophages / Q000166:cytology / Q000201:enzymology*; D051379:Mice; D009154:Mutation; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism*; D010588:Phagosomes / Q000201:enzymology / Q000235:genetics; D018129:Phosphatidylinositol Phosphates / Q000235:genetics / Q000378:metabolism; D016897:Respiratory Burst / Q000502:physiology*; D015398:Signal Transduction / Q000502:physiology*
chemical_list: D004075:Diglycerides; D018129:Phosphatidylinositol Phosphates; C060974:phosphatidylinositol 3,4,5-triphosphate; C055525:phosphatidylinositol 3-phosphate; D019255:NADPH Oxidases; C480281:DAGLB protein, human; D008071:Lipoprotein Lipase; C577904:diacylglycerol lipase beta, mouse
keywords: Diacylglycerol; Macrophages; NADPH Oxidase; Phagocytosis; Phosphatidic Acid